Inhibition studies of glucose-6-phosphate dehydrogenase from tetracycline-producing Streptomyces aureofaciens

NAD-linked activity of glucose-6-phosphate dehydrogenase from both low-producing and high-producing strains of Streptomyces aureofaciens was inhibited by ATP, ADP, AMP and Pi. The inhibition constants indicate that ADP was the most potent inhibitor. The NADP-linked activity remained unaffected even at relatively high concentrations of these inhibitors. All inhibitions of the NAD-linked activity were competitive with respect to NAD and noncompetitive with respect to glucose-6-phosphate. The results represent a possible new regulatory mechanism of glucose-6-phosphate dehydrogenase from a streptomycete and emphasize its involvement in the regulation of the biosynthesis of tetracyclines.     

Kinetic properties of human placental glucose-6-phosphate dehydrogenase

The kinetic properties of placental glucose-6-phosphate dehydrogenase were studied, since this enzyme is expected to be an important component of the placental protection system. In this capacity it is also very important for the health of the fetus. The placental enzyme obeyed "Rapid Equilibrium Ordered Bi Bi" sequential kinetics with K(m) values of 40+/-8 microM for glucose-6-phosphate and 20+/-10 microM for NADP. Glucose-6-phosphate, 2-deoxyglucose-6-phosphate and galactose-6-phosphate were used with catalytic efficiencies (k(cat)/K(m)) of 7.4 x 10(6), 4.89 x 10(4) and 1.57 x 10(4) M(-1).s(-1), respectively. The K(m)app values for galactose-6-phosphate and for 2-deoxyglucose-6-phosphate were 10+/-2 and 0.87+/-0.06 mM. With galactose-6-phosphate as substrate, the same K(m) value for NADP as glucose-6-phosphate was obtained and it was independent of galactose-6-phosphate concentration. On the other hand, when 2-deoxyglucose-6-phosphate used as substrate, the K(m) for NADP decreased from 30+/-6 to 10+/-2 microM as the substrate concentration was increased from 0.3 to 1.5 mM. Deamino-NADP, but not NAD, was a coenzyme for placental glucose-6-phosphate dehydrogenase. The catalytic efficiencies of NADP and deamino-NADP (glucose-6-phosphate as substrate) were 1.48 x 10(7) and 4.80 x 10(6) M(-1)s(-1), respectively. With both coenzymes, a hyperbolic saturation and an inhibition above 300 microM coenzyme concentration, was observed. Human placental glucose-6-phosphate dehydrogenase was inhibited competitively by 2,3-diphosphoglycerate (K(i)=15+/-3 mM) and NADPH (K(i)=17.1+/-3.2 microM). The small dissociation constant for the G6PD:NADPH complex pointed to tight enzyme:NADPH binding and the important role of NADPH in the regulation of the pentose phosphate pathway.     

Kinetic properties of normal human erythrocyte glucose-6-phosphate dehydrogenase dimers.

The steady-state kinetics of normal human erythrocyte glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49) dimers were studied as a function of pH and temperature. Inhibition studies using glucosamine 6-phosphate, NADPH and p-hydroxymercuribenzoate (P-OHMB) were also carried out at pH 8.0. The existence of two binding sites on the enzyme with a transition from low to high affinity for NADP+ when NADP+ concentration is increased is indicated by the nonlinear Lineweaver-Burk plots and sigmoid kinetic patterns. NADPH inhibition was found to be competitive with respect to NADP+ and non-competitive with respect to glucose-6-phosphate. Logarithmic plot of Vmax against pH and inactivation by P-OHMB indicate the participation in the reaction mechanism of imidazolium group of histidine and sulhydryl groups. The initial velocity and product inhibition data gave results which are consistent with the dimeric enzyme following an ordered sequential mechanism. A possible random mechanism is ruled out by the inhibition results of glucosamine 6-phosphate.     

Kinetic properties of glucose-6-phosphate dehydrogenase from lamb kidney cortex

Glucose-6-phosphate dehydrogenase is the key regulatory enzyme of the pentose phosphate pathway and one of the products of this enzyme; NADPH has a critical role in the defence system against the free radicals. In this study, glucose-6-phosphate dehydrogenase from lamb kidney cortex kinetic properties is examined. The purification procedure is composed of two steps after ultracentrifugation for rapid and easy purification: 2', 5'-ADP Sepharose 4B affinity and DEAE Sepharose Fast Flow anion exchange chromatography. Previously, we used this procedure for the purification of glucose-6-phosphate dehydrogenase from bovine lens. The double reciprocal plots and product inhibition studies showed that the enzyme obeys 'Ordered Bi Bi' mechanism: K(m NADP+)K(m G-6-P) and K(i G-6-P) (dissociation constant of the enzyme--G-6-P complex) were found to be 0.018 +/- 0.002, 0.039 +/- 0.006 and 0.029 +/- 0.005 mM, respectively, by using nonlinear regression analysis. The enzyme was stable at 4 degrees C for a week.     

Kinetic studies on the hydrogen peroxide elimination by cultured PC12 cells: rate limitation by glucose-6-phosphate dehydrogenase

Long chain acyl-Coenzyme A esters (acyl-CoAs) are key substrates in many enzymic reactions of lipid metabolism. Due to their amphiphilic nature, the membrane localization of these molecules cannot be established by subcellular membrane fractionation and usual biochemical studies. We have developed another approach based on ultrastructural immunogold cytochemistry. To preserve the acyl-CoA membrane content, the plant material was freeze substituted and cryoembedded after short aldehyde fixation followed by quick freezing. Using Arabidopsis thaliana root cells and specific antibodies raised against acyl-CoAs, we show that acyl-CoAs are mainly localized in endoplasmic reticulum membranes.Our results demonstrate the value of cryo-methods for the accurate localization of labile metabolites in plant cells.     

Inhibition of lipid synthesis and glucose-6-phosphate dehydrogenase in rat skin by dehydroepiandrosterone

Lipid synthesis from acetate-1-(14)C by rat skin was inhibited 44-56% by 2.5 x 10(-4) m dehydroepiandrosterone (DHA) in vitro with or without addition of glucose in the incubation medium. This inhibition affected all the lipid fractions examined (hydrocarbons, sterols, sterol esters, tri-, di- and monoglycerides, fatty acids, and polar lipids) and could be reversed by NADPH. DHA also inhibited lipid synthesis from glucose-U-(14)C and the formation of (14)CO(2) from glucose-1-(14)C, indicating interference with pentose cycle activity. Experiments with the 105,000 g supernatant fluid of rat skin homogenates demonstrated considerable activities of malic enzyme (ME) (12.6 nmoles of NADPH generated per min per mg of protein), of glucose-6-phosphate dehydrogenase (G6PD), and of 6-phosphogluconate dehydrogenase (6PGD) (17.5 nmoles of NADPH generated per min per mg of protein). G6PD was inhibited 98% by 2.5 x 10(-4) m dehydroepiandrosterone, while 6PGD and ME were not affected. It can be estimated from these data that the pentose cycle may contribute 41-57% of the NADPH needed for lipid synthesis in rat skin; the remainder of the necessary NADPH is presumably supplied by malic enzyme.     

Drug-induced haemolysis in glucose-6-phosphate dehydrogenase deficiency.

People with the variants of glucose-6-phosphate dehydrogenase (GPD) deficiency common in the southern Chinese (Canton, B(-)Chinese, and Hong Kong-Pokfulam) have a moderate shortening of red-cell survival but no anaemia when they are in the steady state. With a cross-transfusion technique, primaquine, nitrofurantoin, and large doses of aspirin were found to aggravate the haemolysis while sulphamethoxazole did so only in some people. Individual differences in drug metabolism may be the reason for this. Many commonly used drugs reported to accentuate haemolysis in GPD deficiency did not shorten red-cell survival.